Characterization of histones from beef pancreas]

The total histone of ox pancreas was fractionated by electrophoresis on 10 and 25 cm polyacrylamide gels according to Panyim and Chalkley (1969). The presence of an additional subfraction within the lysine rich histone was stated. In the course of the fractionation of total histone according to the method of Oliver et al. (1972) this additional histone component was extracted together with F1 histone.     

Tertiary structure in N-linked oligosaccharides

Distance constraints derived from two-dimensional nuclear Overhauser effect measurements have been used to define the orientation of the Man alpha 1-3Man beta linkage in seven different N-linked oligosaccharides, all containing the common pentasaccharide core Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. Conformational invariance of the Man alpha 1-3Man beta linkage was found for those structures bearing substitutions on the Man alpha 1-3Man beta antenna. However, the presence of either a GlcNAc residue in the beta 1-4 linkage to Man beta ("bisecting GlcNAc") or a xylose residue in the beta 1-2 linkage to Man beta of the trimannosyl core was found to generate conformational transitions that were similar. These transitions were accompanied by characteristic chemical shift perturbations of proton resonances in the vicinity of the Man alpha 1-3Man beta linkage. Molecular orbital energy calculations suggest that the conformational transition between the unsubstituted and substituted cores arises from energetic constraints in the vicinity of the Man alpha 1-3Man beta linkage, rather than specific long-range interactions. These data taken together with our previous results on the Man alpha 1-6Man beta linkage [Homans, S. W., Dwek R. A., Boyd, J., Mahmoudian, M., Richards, W. G., & Rademacher, T. W. (1986) Biochemistry 25, 6342] allow us to discuss the consequences of the modulation of oligosaccharide solution conformations.     

Properties of histones on hydrophobic chromatography]

The hydrophobic chromatography on alkylated Sepharose allows to separate the histones into three groups which exhibit an increasing affinity for the support HI less than H2A-H2B less than H3-H4. In this fractionation procedure, the behaviour of the histones taken separately or pair-associated, is discussed in relation with the ability of these proteins to complex each other in vitro and in vivo.     

Tertiary structure and function of transfer-RNA

Summary In analogy to the globular protein structures the predominant stabilization factor of which is surface energy a compact model with the smallest possible surface of the tertiary structure of t-RNA is suggested. The principle of folding is — according to the clover leaf model — the superposition of the three leaves. The three loops form a ring (loop-stack). The stem can be placed on this molecular body bringing the CCA end into the vicinity of the loop-stack, where the CCA end can be bound. The structures of the RNA chain are subordinated to this globular structural principle. Under the influence of the sequence the CCA end is able to undergo structural changes, thus allowing to explain the role of t-RNA in the aminoacylation process and the structural differences between t-RNA, aminoacyl-t-RNA, and peptidyl-t-RNA. Furthermore it is possible to comment on the existence of the characteristic and modified bases, the role of magnesium ions and the chemical reactions of these compounds.I am particularly greatful to Dr. P. Fromageot, Dr. W. Guschlbauer (Saclay), Prof. Dr. R. Brunner, and Dr. M. Röhr (Vienna) for helpful discussions. Dr. J. Catlin helped me in the preparation of this paper. I was supported in this work by a Joliot-Curie fellowship.     

Tertiary structure of bacterial selenocysteine tRNA

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA^Sec) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA^Sec from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA^Sec assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA^Secs, A. aeolicus tRNA^Sec has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA^Sec in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA^Sec. Similar interactions exist in the reported tRNA^Ser and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA^Sec in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA^Sec to enter the SerRS catalytic site.     

Tertiary structure prediction of SARS coronavirus helicase

SARS coronavirus, SCV, has been recently responsible of a sudden and widespread infection which caused almost 800 victims. The limited amount of SCV protein structural information is partially responsible of the lack of specific drugs against the virus. Coronavirus helicases are very conserved and peculiar proteins which have been proposed as suitable targets for antiviral drugs, such as bananins, which have been recently shown to inhibit the SCV helicase in vitro. Here, the quaternary structure of SCV helicase has been predicted, which will provide a solid foundation for the rational design of other antiviral helicase inhibitors.     

pH effects on the structure of the inner histones

The structure of the inner histone complex extracted from chicken erythrocyte chromatin with 2 M NaCl has been studied as a function of pH. At pH 6, the complex dissociates to (H3–H4)₂ tetramer and H2A·H2B dimer, with little change in α-helix content (as monitored by circular dichroism at 222 nm). Although the circular dichroism of tyrosyl side chains is also largely unchanged by the dissociation, measurements of intrinsic fluorescence do suggest a change in the environment of one or more tyrosines as a result of dissociation. Below pH 4, the histones become partially unfolded, lose specific secondary and tertiary structure, and undergo nonspecific aggregation. Both the pH 6 and 4 transitions, which are largely reversible, parallel pH-induced structural changes of nucleosomes (Zama, M., Olins, D.E., Prescott, B. and Thomas, G.J. (1978) Nucleic Acids Res. 5, 3881–3897). The results are consistent with the presence of tyrosine residues at the histone subunit-subunit contacts and suggest that histone conformation within the globular regions is largely independent of histone-DNA interactions.     

Effects of histones on phagocytosis and phagocytic metabolism]

Enhanced phagocytosis and no intracellular glycogen breakdown was found in guinea-pig peritoneal polymorphonuclear leukocytes (PMN) incubated together with heat-killed staphylococci (Twort) treated with a calf thymus histone fraction (P II), for 1 hr at 37 degrees C. Glcose consumption and lactate production of these PMN's were as high as in phagocytosis of the same germ untreated with P II, and their oxygen consumption was depressed significantly.     

Tertiary structure of Staphylococcus aureus cell wall murein

The recently described scaffold model of murein architecture depicts the gram-negative bacterial cell wall as a gel-like matrix composed of cross-linked glycan strands oriented perpendicularly to the plasma membrane while peptide bridges adopt a parallel orientation (B. A. Dmitriev, F. V. Toukach, K. J. Schaper, O. Holst, E. T. Rietschel, and S. Ehlers, J. Bacteriol. 185:3458-3468, 2003). Based on the scaffold model, we now present computer simulation studies on the peptidoglycan arrangement of the gram-positive organism Staphylococcus aureus, which show that the orientation of peptide bridges is critical for the highly cross-linked murein architecture of this microorganism. According to the proposed refined model, staphylococcal murein is composed of glycan and oligopeptide chains, both running in a plane that is perpendicular to the plasma membrane, with oligopeptide chains adopting a zigzag conformation and zippering adjacent glycan strands along their lengths. In contrast to previous models of murein in gram-positive bacteria, this model reflects the high degree of cross-linking that is the hallmark of the staphylococcal cell wall and is compatible with distinguishing features of S. aureus cytokinesis such as the triple consecutive alteration of the division plane orientation and the strictly centripetal mode of septum closure.     

Age-related peculiarities of acetylation of rat liver nuclear histones]

The intensity of acetylation of different histone fraction is found to change in postnatal ontogenesis of albino rats. The intensity of acetylation of lisine-rich histone (F1) was maximal in 3-and 12-month old animals. The label incorporation into F3 (arginine-rich) histones considerably decreased in the time interval between 1st and 3d months while its incorporation increased into F2 histones furing all the period of postnatal development studied.     

Association of proteinases with histones from rat thymus nuclei]

Histone H2A, H2B, and H1--specific proteinases tightly associated with histones were shown to be present in rat thymus nuclei. The activity of proteinases tightly associated with histones increases after exposure of animals to gamma-rays. The denatured DNA activated the histone H1-specific proteinase. These proteinase dissociated from histones in the presence of dithiothreitol. The histones and proteinases were divided into fractions by chromatography on DEAE-cellulose in the presence of 5 mM dithiothreitol.     

Studies on DNA bound to histones in chromatin]

Regions of DNA protected by histones against the action of DNAse 1 in the chromatin were isolated. Such DNA fragments (subhistones DNA) have 80% double helix structure, their nucleotide composition is close to that of total DNA, and their sedimentation constant is within the range of 2-2.7S for completely denatured molecules. Kinetics of renaturation of subhistone DNA was studied: within a wide range of Cot values, renaturation curves of total and subhistone DNA are almost identical. According to the data on hybridization with nuclear d-RNA, subhistone DNA is transcribed in the cell. The data obtained witness for uniform character of distribution of histones along the DNA chain in the chromatin. DNA sites which are active in RNA synthesis seem to be bound to histones as well as the non-active ones. No significant difference was found in the hybridization of subhistone DNA from rat liver and thymus with ibver nuclear RNA.     

Tertiary structure stabilization promotes hairpin ribozyme ligation.

The hairpin ribozyme catalyzes a reversible RNA cleavage reaction that participates in processing intermediates of viral satellite RNA replication in plants. A minimal hairpin ribozyme consists of two helix-loop-helix segments. These segments associate noncoaxially in the active folded structure in a way that brings catalytically important loop nucleotides into close proximity. The hairpin ribozyme in the satellite RNA of Tobacco Ringspot Virus assembles in the context of a four-way helical junction. Recent physical characterization of hairpin ribozyme structures using fluorescence resonance energy transfer demonstrated enhanced stability of the folded structure in the context of a four-way helical junction compared to minimal hairpin ribozyme variants. Analysis of the functional consequences of this modification of the helical junction has revealed two changes in the hairpin ribozyme kinetic mechanism. First, ribozymes with a four-way helical junction bind 3' cleavage products with much higher affinity than minimal hairpin ribozymes, evidence that tertiary interactions within the folded structure contribute to product binding energy. Second, the balance between ligation and cleavage shifts in favor of ligation. The enhanced ligation activity of hairpin ribozymes that contain a four-way helical junction supports the notion that tertiary structure stability is a major determinant of the hairpin ribozyme proficiency as a ligase and illustrates the link between RNA structure and biological function.     

Tertiary structure of bacterial murein: the scaffold model

Although the chemical structure and physical properties of peptidoglycan have been elucidated for some time, the precise three-dimensional organization of murein has remained elusive. Earlier published computer simulations of the bacterial murein architecture modeled peptidoglycan strands in either a regular (D. Pink, J. Moeller, B. Quinn, M. Jericho, and T. Beveridge, J. Bacteriol. 182: 5925-5930, 2000) or an irregular (A. Koch, J. Theor. Biol. 204: 533-541, 2000) parallel orientation with respect to the plasma membrane. However, after integrating published experimental data on glycan chain length distribution and the degree of peptide side chain cross-linking into this computer simulation, we now report that the proposed planar network of murein appears largely dysfunctional. In contrast, a scaffold model of murein architecture, which assumes that glycan strands extend perpendicularly to the plasma membrane, was found to accommodate published experimental evidence and yield a viable stress-bearing matrix. Moreover, this model is in accordance with the well-established principle of murein assembly in vivo, i.e., sequential attachment of strands to the preexisting structure. For the first time, the phenomenon of division plane alternation in dividing bacteria can be reconciled with a computer model of the molecular architecture of murein.